Cold plasma electroporation of medication and associated methods

ABSTRACT

A method and device to apply a cold plasma to a substance at a treatment surface of a patient to cause electroporation of the substance into cells of the patient. The substance can be previously applied to the treatment surface. Alternatively, the substance can be placed in a foam-like material within a tip that passes the cold plasma from the cold plasma device to the treatment area. The tip can be a cannula device with an aperture at the distal end. The cannula device can also have apertures along a portion of the length of the cannula device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/145,320, filed Dec. 31, 2013, which claims the benefit of U.S.Provisional Application No. 61/747,871, filed Dec. 31, 2012 and entitled“Cold Plasma Electroporation of Medication and Associated Methods,” allof which are incorporated herein by reference in their entirety.

This application is related to U.S. Provisional Application No.60/913,369, filed Apr. 23, 2007; U.S. patent application Ser. No.12/038,159, filed Feb. 27, 2008 (which issued as U.S. Pat. No.7,633,231); U.S. patent application Ser. No. 13/620,118, filed Sep. 14,2012; and U.S. patent application Ser. No. 13/620,236, filed Sep. 14,2012, each of which are herein incorporated by reference in theirentireties.

BACKGROUND

Field of the Art

The present invention relates to devices and methods for cold plasmamedical treatment, and, more particularly, to such devices and methodsfor cold plasma electroporation of medications and bioactive agents intocells.

Background Art

Cold plasmas (i.e., non-thermal plasmas) are produced by the delivery ofpulsed high voltage signals to a suitable electrode. Cold plasma devicesmay take the form of a gas jet device or a dielectric barrier discharge(DBD) device.

Electroporation is the process of exposing cells to electrical fields,as illustrated in FIG. 1. When a biological cell 180 is exposed toprogrammed electric pulses from electrodes 170, the lipid membrane ofthe cell can be altered and become permeable 160. The change in the cellmembrane may be of a plastic (temporary) or permanent nature, and thesechanges are commonly referred to as reversible or irreversiblepermeabilization, respectively.

One of the primary reasons to electroporate a cell, or group of cells,is to transport a molecule across the membrane that otherwise would beunable to cross this barrier, or would require cellular energy topump/transport in the absence of applied energy. Thereforeelectroporation allows the cell membrane to become permeablized, and isfrequently used to either insert proteins 110 into the cell membrane,introduce large 130 or small 120 molecules into the cell(s), inducecellular fusion 140, or to destroy the cell membrane 150 altogether.

Irreversible premeabilization can permanently damage a cell and lead toapoptosis or other mechanisms of cell death. Controllable apoptosis hasbeen used in biofouling control, debacterialization, and drug-freecancer therapies.

Reversible electroporation is primarily used as a method of moleculardelivery, transferring a wide array of molecules, such as drugs, ions,dyes, tracers, oligonucleotides, RNA, antibodies, proteins, etc., intoand out of cells. There are several advantages to usingelectroporation-moderated molecular delivery over conventional methods.Electroporation is generally non-invasive, drug free, non-toxic andrapidly accomplished. Due to the fact that electroporation is a physicalprocess between the supplied electric field and the cell membrane, it isless influenced by the specific cell type when compared to conventionalmethods.

Electroporation is demonstrably effective in both in vivo and in vitroclinical studies and applications, and has been employed for treatingvarious cancers including lung, skin, breast, leukemia, specific bonecancers, and for DNA vaccination.

BRIEF SUMMARY OF THE INVENTION

An embodiment is described of a method of applying a substance to atreatment area of a patient. The method also includes applying a coldplasma from a cold plasma device to the substance for a predeterminedtreatment time to thereby cause electroporation of the substance intocells of the patient.

A further embodiment is described of a method of generating a coldplasma from a cold plasma device. The method also includes passing thecold plasma from the cold plasma device via a nozzle to a treatment areaof a patient for a predetermined treatment time. The nozzle includes anelement (e.g., disk) positioned in the nozzle, the element (e.g., disk)including a substance. The passing of the cold plasma through thesubstance thereby causes electroporation of the substance into cells ofthe patient.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 illustrates a number of applications of electroporation.

FIG. 2 illustrates an exemplary tip assembly that includes a porous foamelement, in accordance with an embodiment of the present invention.

FIGS. 3A and 3B illustrate different tips for use with a cold plasmageneration device, in accordance with an embodiment of the presentinvention.

FIG. 4 illustrates a cannula tube embodiment that includes a porous foamelement, in accordance with an embodiment of the present invention.

FIG. 5 illustrates a method for electroporation using a cold plasma, inaccordance with an embodiment of the present invention.

FIG. 6 illustrates a method for electroporation using a cold plasma, inaccordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Cold temperature plasmas have attracted a great deal of enthusiasm andinterest by virtue of their provision of plasmas at relatively low gastemperatures. The provision of plasmas at such a temperature is ofinterest to a variety of applications, including wound healing,anti-bacterial processes, various other medical therapies andsterilization. As noted earlier, cold plasmas (i.e., non-thermalplasmas) are produced by the delivery of pulsed high voltage signals toa suitable electrode. Cold plasma devices may take the form of a gas jetdevice or a dielectric barrier discharge (DBD) device. In the context ofthis application, the methods disclosed herein can be used with anyplatform for the generation of cold plasma. Accordingly, the methods arenot limited to the use of a DBD device, a gas jet device, or a coldplasma generated using a multi-frequency harmonic-rich power supply.

FIG. 1 illustrates a number of applications of electroporation (FIG. 1has been adapted from material presented in a March 2008 lecture by Dr.Damijan Miklavcic. See http://videolectures.net/tict08_miklavcic_ebt/).The main use of electroporation-based technologies and treatments inmodern medicine and industrial applications are: molecular cell biologyresearch, cell fusion, gene expression silencing by small interferingRNA (siRNA), electrochemotherapy, protein insertion into a cellmembrane, gene therapy based on electro-gene transfer, variousapplications within biotechnology, tissue ablation, cell fusion formonoclonal antibody production, water and liquid food sterilization, andtransdermal drug delivery.

Electrochemotherapy is the combination of chemotherapy andelectroporation during which an electric pulse generator is used toapply an electrical current through electrodes that are inserted intothe body on either side of a cancerous tumor. A chemotherapy drug isthen injected near the tumor site such that the chemotherapeuticsurrounds the cell. Once the electric pulse is applied from thegenerator through the electrodes, the increase in cell membranepermeability allows access to the cytosol (intracellular fluid). If thepulsed current amplitude and duration is carefully moderated, then thepores of the cells can reseal (reversible electroporation) encapsulatingthe chemotherapeutic. A similar method might be employed withantibiotics and bacterium.

Current methods of electroporation in the application of vitalmedications, such as during electrochemotherapy, require the electrodesto be inserted into the patient, in addition to the physicalintroduction of the chemotherapeutic. These procedures can be painful,add extra steps and complexity to the treatment protocol, and are apotential source of infection transmission. Significant collateral celldeath and low delivery efficiency have challenged traditional methods ofelectroporation (Andre, F. M., et al., 2010).

A commonly applied technique for drug delivery through the skin withoutthe use of an injection needle is iontophoresis. Iontophoresis, alsoknown as electromotive drug administration (EMDA), uses a relativelysmall electric charge to deliver a medication, a chemical agent, or abioactive agent through a patient's skin. While traditional methods ofinducing iontophoresis can be effective in specific circumstances, it istime consuming to administer, can create tingling, irritation or burningin the patient, has a markedly lower efficacy with nonpolar drugs, andrequires an intact stratum corneum (outermost layer of the epidermis)for effective drug penetration, which means that it cannot be used ondamaged skin. In the context of this disclosure, the word“electroporation” is used to include iontophoresis.

The cold plasma method (including, but not limited to, a multi-frequencyharmonic-rich cold plasma treatment) of transdermal electroporation ofmedication is simple, painless, and an effective method of generatingelectroporation for the successful introduction of a multitude ofmedications or bioactive agents to a patient's body or cells. In U.S.Non-Provisional application Ser. No. 13/620,236, filed on Sep. 14, 2012,specific tips are described that are designed to produce plumes ofplasma where the delivery of biological materials and agents (viralvectors, DNA, etc.), chemicals or drugs, and proteins in addition to theplasma itself is possible when desirable. The disclosure of U.S. patentapplication Ser. No. 13/620,236, filed Sep. 14, 2012, is included hereinby reference in its entirety.

The prescribed biological materials and agents (viral vectors, DNA,etc.), chemicals or drugs, and/or proteins can be introduced into thepatient's body in one of three main cold plasma techniques ormethodologies, as described herein. First and simplest, the prescribedmaterial (in liquid, gel, or powdered form) can be applied to theepidermis of the recipient (FIG. 3A and FIG. 5). In this embodiment, theprescribed agent can be lidocaine, ropivacaine, bupivacaine, Marcaine®,or another anesthetic, and the treatment time to achieve therapeuticpenetration of the substrate can be 30, 60, or 300 seconds. In analternative embodiment, the prescribed agent can be an antibiotic with atreatment time of 30, 60, or 300 seconds. In a further alternativeembodiment, the prescribed agent can be a chemotherapeutic agent with atreatment of 30, 60, 300, or 600 seconds. The above details are merelyexemplary, and not limiting in scope.

A pulsed electrical energy source generates a cold plasma, and the coldplasma, carries a pulsed electrical energy field. When the cold plasmais directed over a recently applied prescribed material, one resultingeffect is a controllable state of electroporation in the cells of thetarget substrate. The consequential increase in cell membranepermeability permits the transfer of the drug or chemical into thetarget cells.

FIG. 2 illustrates a second embodiment of the present invention.Referring to FIG. 2, an expanded graphical view of a 42 mm circumferencetip is illustrated for use with a cold plasma generation device. Moregenerally, apertures within the tip may be of any shape, includingcircular, slit and polygon shapes. Included in the tip is a porous foam,made from a material such as a porous foam or other suitable materialsknown to one skilled in the art, that is inserted within the assembly.The foam provides a potential carrying mechanism for the inclusion ofwater, solutions or drugs for introduction into the cold plasma stream,which are then electroporated into the tissues. With the addition of aporous foam element to any of the cold plasma tips (as illustrated inFIG. 2), this treatment method applies the appropriate prescribedbiological materials and agents (viral vectors, DNA, etc.), chemicals ordrugs, and/or proteins to the porous element, with the result that thepatient is treated with the non-thermal plasma for a combined therapy.The prescribed material contained within the porous element would betransported in the cold plasma plume to the target treatment substrateand thereby introduced into the cytosol of the target cells via theelectroporation induced by the cold plasma.

FIGS. 3A and 3B illustrate different tips 310, 320 that can be affixedto the outlet port of a cold plasma device. These tips representexamples of different methods of administering a prescribed material toa patient. For example, for the embodiment illustrated in FIG. 3A,prescribed biological materials and agents (viral vectors, DNA, etc.),chemicals or drugs, and/or proteins are first applied to the treatmentarea on the patient's skin. Next, plasma plume 312 is applied to thetreatment area to induce electroporation and thereby deliver the agentor drug to the cells of the patient.

FIG. 3B illustrates an alternative approach, whereby illustrated tip 320includes a porous foam element, 322, that can be added to any of thecold plasma tips in order to introduce a prescribed agent into theplasma stream for electroporation applications. In both cases, the tipscan be either reusable or disposable.

FIG. 4 illustrate a third embodiment of the present invention. Referringto FIG. 4, a cannula tube embodiment is illustrated that can deliver anyprescribed agent into a bodily lumen, cavum, vestibule, or buccal cavityvia the porous foam elements 440. In this third method of delivery ofprescribed biological materials and agents (viral vectors, DNA, etc.),chemicals or drugs, and/or proteins in a non-thermal plasma treatmentprotocol, the prescribed material can be added to an open-celled foamelement that is contained within the main lumen of a cannula device. Notall medical treatments can be performed external to the body of a humanor animal. In many cases, the treatment site is internal to a body andaccess to such a site requires the provision of tools that are placed atthe end of various elongated devices, such as laparoscopic, arthroscopicand endoscopic devices.

Continuing to refer to FIG. 4, an exemplary cannula tube 410 can beattached to the outlet port of the cold plasma device. In an exemplaryembodiment, cannula tube 410 has a single aperture 420 at the proximalend that is attached to the outlet port of the cold plasma device.Cannula tube 410 has a length sufficient to reach a desired treatmentarea. Typically, the treatment area is internal to a human being oranimal, where the treatment area is accessible via an opening such asmouth, nose, arterial or venal entry point, or transdermally through aport (laproscopic, arthroscopic). Thus, a cannula tube can be used forinternal treatment within any bodily lumen, cavum, vestibule or buccalcavity and can be utilized to deliver any appropriate biologicalmaterials and agents (viral vectors, DNA, etc.), chemicals or drugs,and/or proteins via the internal open-celled foam element 440. In anembodiment of the present invention, the cannula tube has a singleaperture 430 at the distal end inserted into the treatment area. Cannulatube 410 can be used for internal treatment within any bodily lumen,cavum, vestibule, or buccal cavity.

In an alternative embodiment (not shown), the exemplary cannula tubeincludes a plurality of apertures at the distal end of the cannula tube,and a porous foam element, which can be enriched with the appropriatechemical or drug substance. In various embodiments, the apertures can beat the end or placed at a variety of locations along a portion of thelength of the cannula tube adjacent to the end of the cannula tube. Inone of these embodiments, the distal end of the cannula tube can besealed, with one or more apertures located along the body length.Cannula tube can be used for internal treatment along the length of anybodily lumen, cavum, vestibule, or buccal cavity. The placement ofopen-celled foam element 440 illustrated in FIG. 4 is merely exemplaryand not limiting to the scope of various embodiments of the presentinvention. For example, the placement of a suitable element may beplaced in any location consistent with delivery of the biologicalmaterials and agents (viral vectors, DNA, etc.), chemicals or drugs,and/or proteins at the desired treatment area. In particular, a suitableelement may be placed at a location that is also driven by ease ofmanufacture, or other considerations. As an example, an element may beplaced at the base of the cannula nozzle, where it attaches to the coldplasma applicator device. Further, the shape of the element can take onany form consistent with its placement at any suitable location of thecannula tube.

The type of material noted in open-celled foam element 440 (asillustrated in FIG. 4) is also merely exemplary of the materials thatcan be used, and not limiting to the scope of various embodiments of thepresent invention. For example, any material that can act as a reservoirof biological materials and agents (viral vectors, DNA, etc.), chemicalsor drugs, and/or proteins while permitting movement of the cold plasmathrough the material falls within the scope of various embodiments ofthe present invention.

In addition, various embodiments of the present invention are notlimited to a particular cold plasma generation approach. For example,embodiments of the present invention may include cold plasma generationapproaches, as well as multi-frequency harmonic-rich cold plasmageneration approaches. In addition, different techniques of cold plasmageneration are also included. For example, cold plasma generation caninclude gas-fed plasma generation devices that take as an input a sourceof an appropriate working gas (e.g., helium or any other suitable gas)and a source of high voltage electrical energy, and output a plasmaplume. Previous work by the inventors in this research area has beendescribed in U.S. Provisional Patent Application No. 60/913,369, U.S.Non-provisional application Ser. No. 12/038,159 (that has issued as U.S.Pat. No. 7,633,231) and the subsequent continuation applications (“the'369 application family”). Different high voltage power supplies mayalso be used to provide the resulting cold plasma (for example, but notlimited to, a multi-frequency harmonic-rich supply as described in the'369 application family, and in U.S. patent application Ser. No.13/620,118, filed Sep. 14, 2012, which is incorporated herein byreference in its entirety). As noted previously, in the context of thisapplication, the methods disclosed herein can be used with any platformfor the generation of cold plasma. Accordingly, the methods are notlimited to the use of a DBD device, a gas jet device, or a cold plasmagenerated using a multi-frequency harmonic-rich power supply.

Devices, other than the gas-fed cold plasma generation device describedabove, can also generate cold plasma. For example, cold plasma can alsobe generated by a dielectric barrier discharge device, which relies on adifferent process to generate the cold plasma. A dielectric barrierdischarge (DBD) device contains at least one conductive electrodecovered by a dielectric layer. The electrical return path is formed bythe ground that can be provided by the target substrate undergoing thecold plasma treatment or by providing an in-built ground for theelectrode. Energy for the dielectric barrier discharge device can beprovided by a high voltage power supply, such as that mentioned above.More generally, energy is input to the dielectric barrier dischargedevice in the form of pulsed DC electrical voltage to form the plasmadischarge. By virtue of the dielectric layer, the discharge is separatedfrom the conductive electrode and electrode etching and gas heating isreduced. The pulsed DC electrical voltage can be varied in amplitude andfrequency to achieve varying regimes of operation. Any deviceincorporating such a principle of cold plasma generation (e.g., a DBDelectrode device) falls within the scope of various embodiments of thepresent invention.

The above embodiments also facilitate an approach that allows for theadministration of a cold plasma treatment protocol while the patient issimultaneously undergoing treatment with a systemic drug. When asystemic drug is being prescribed to a patient (such as oral orintravenous (IV) antibiotics or chemotherapy), it may be desirable toalso treat a specific area, or multiple areas, with cold plasma. Thecold plasma could be applied with or without chemical agents beingdelivered through electroporation. This combined methodology would allowfor the cold plasma to be used at a specific site to treat an infectionor tumor with the systemic drug treatment method for a cumulativehealing effect.

FIGS. 5 and 6 provide further details of two approaches to the combinedmethodology. Referring to FIG. 5, a methodology is illustrated for usinga cold plasma device for administration of prescribed biologicalmaterials and agents (viral vectors, DNA, etc.), chemicals or drugs,and/or proteins treatment in combination with non-thermal plasma whenthe chemical or drug is applied directly to the patient's skin ortreatment substrate. In step 510, appropriate biological materials andagents (viral vectors, DNA, etc.), chemicals or drugs, and/or proteinsare applied to the treatment substrate. In step 520, if the cold plasmadevice is a gas jet device, an appropriate delivery nozzle may beoptionally attached to the cold plasma treatment device. In step 530,the predetermined cold plasma treatment protocol is applied to thepatient, thereby administering the prescribed drug or chemical directlyinto the target cells of the patient via electroporation delivered bythe cold plasma. Target cells can include the skin, a wound, a surgicalsite, a structure exposed during a surgical procedure, a tumor, or anytissue that can be contacted directly by the combination of thetherapeutic agent and the plasma.

Referring to FIG. 6, a methodology is illustrated for using a coldplasma device for administration of the prescribed biological materialsand agents (viral vectors, DNA, etc.), chemicals or drugs, and/orproteins that are applied to a porous foam element. The prescribedmaterials are introduced to the target cells via electroporation throughadministration of a non-thermal plasma treatment protocol with a coldplasma device

In step 610, appropriate biological materials and agents (viral vectors,DNA, etc.), chemicals or drugs, and/or proteins are applied to theporous foam element. In step 620, an appropriate delivery nozzlecontaining the treated porous foam element is attached to the coldplasma treatment device. In step 630, the predetermined cold plasmatreatment protocol is applied to the patient, thereby administering theprescribed drug or chemical into the target cells of the patient viaelectroporation delivered by the plasma.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. A method, comprising: generating a cold plasmafrom a cold plasma device; and passing the cold plasma from the coldplasma device through a body entry point and via a cannula tube to atreatment area of a patient for a predetermined treatment time, whereinthe cannula tube comprises an element positioned in the cannula tube,the element comprising a chemical-or-drug substance and an open-celledfoam material configured to absorb the chemical-or-drug substance, andpassing the cold plasma through the chemical-or-drug substance therebycauses electroporation of the chemical-or-drug substance into cells of apatient.
 2. The method of claim 1, wherein the chemical-or-drugsubstance comprises at least one of a biological material, agent, viralvector, DNA, chemical, drug, or protein.
 3. The method of claim 1,wherein the chemical-or-drug substance comprises at least one oflidocaine, ropivacaine, bupivacaine and an anesthetic.
 4. The method ofclaim 1, wherein the chemical-or-drug substance comprises achemotherapeutic agent.
 5. The method of claim 1, wherein thepredetermined treatment time and the chemical-or-drug substance areassociated with a treatment protocol.
 6. The method of claim 1, whereinthe body entry point is a mouth or a nose.
 7. The method of claim 1,wherein the body entry point is an arterial entry point or a venal entrypoint.
 8. The method of claim 1, wherein the body entry point is atransdermal entry point obtained using a laproscope or an arthroscope.9. The method of claim 1, wherein the treatment area is within one of abodily lumen, a cavum, a vestibule, or a buccal cavity.
 10. The methodof claim 1, wherein the cannula tube comprises an aperture adjacent toan end of the cannula tube.
 11. The method of claim 1, whereingenerating the cold plasma from the cold plasma device comprisesgenerating the cold plasma from a multi-frequency harmonic-rich coldplasma generator.
 12. The method of claim 1, wherein generating the coldplasma from the cold plasma device comprises generating the cold plasmafrom a dielectric barrier discharge (DBD) cold plasma generator.
 13. Themethod of claim 1, wherein generating the cold plasma from the coldplasma device comprises generating the cold plasma from a gas jet coldplasma generator.
 14. The method of claim 1, wherein the element islocated at a base of the cannula tube.
 15. The method of claim 1,wherein the cannula tube comprises a plurality of apertures adjacent toan end of the cannula tube.
 16. The method of claim 1, wherein thecannula tube comprises a plurality of apertures located along a portionof the cannula tube.
 17. The method of claim 1, wherein the treatmentarea is a surgical site.
 18. The method of claim 1, wherein thetreatment area includes a tumor.
 19. The method of claim 1, wherein thetreatment area includes a structure exposed during a surgical procedure.20. The method of claim 1, wherein the treatment area includes a wound.